DNA sequence data for many human exomes and genomes are accumulating at an ever-increasing pace, yet an understanding of the effect of small genetic variations on protein function lags behind. We have developed a method to analyze the function of up to ~1 million variants of a protein using next generation DNA sequencing and protein display formats that link genotype to phenotype. Our work to date has used small protein domains (typically <100 amino acids) and functional assays in yeast or in vitro. However, advances in technology suggest that we should be able to extend our approach to much larger proteins and more complex assays, in particular assays in human cells. Our overall goal is to develop the technology to rapidly assess the function, in human cells, of all th variants of a large human protein that has multiple activities and interactions. This technology will be prototyped using the BRCAl protein - in which germ-line mutations result in a vastly increased risk of breast and ovarian cancer-and then extended to other proteins implicated in cancer risk. We will compare the results from our assays to the data on disease risk and progression for known variants in order to establish the utility of our high throughput approach. Our Driving Biomedical Project will employ a DNA repair assay in human cells to analyze the activity of BRCAl variants. Our specific aims are: 1) To generate all possible single amino acid changes in the BRCAl protein and to assess the variants for their proficiency in DNA repair (using the whole protein) and in E3 ligase activity (using a 304 amino acid domain); 2) To compare the quantitative fitness of the BRCA1 variants obtained in our assays with a database of disease alleles; 3) To extend the approach to other human genes relevant to cancer and amenable to similar assays, such as BRCA2, BARD1 and CHEK2.